Optimization of transplantation methods using isolated mesenchymal stem/stromal cells: clinical trials of inflammatory bowel diseases as an example

Mesenchymal stem/stromal cells (MSCs) are distributed in various tissues and are used in clinical applications as a source of transplanted cells because of their easy harvestability. Although MSCs express numerous cell-surface antigens, single-cell analyses have revealed a highly heterogeneous cell population depending on the original tissue and donor conditions, including age and interindividual differences. This heterogeneity leads to differences in their functions, such as multipotency and immunomodulatory effects, making it challenging to effectively treat targeted diseases. The therapeutic efficacy of MSCs is controversial and depends on the implantation site. Thus, there is no established recipe for the transplantation of MSCs (including the type of disease, type of origin, method of cell culture, form of transplanted cells, and site of delivery). Our recent preclinical study identified appropriate MSCs and their suitable transplantation routes in a mouse model of inflammatory bowel disease (IBD). Three-dimensional (3D) cultures of MSCs have been demonstrated to enhance their properties and sustain engraftment at the lesion site. In this note, we explore the methods of MSC transplantation for treating IBDs, especially Crohn’s disease, from clinical trials published over the past decade. Given the functional changes in MSCs in 3D culture, we also investigate the clinical trials using 3D constructs of MSCs and explore suitable diseases that might benefit from this approach. Furthermore, we discuss the advantages of the prospective isolation of MSCs in terms of interindividual variability. This note highlights the need to define the method of MSC transplantation, including interindividual variability, the culture period, and the transplantation route.


Background
The number of patients with inflammatory bowel diseases (IBDs), including ulcerative colitis (UC) and Crohn's disease (CD), was approximately 5 million worldwide in 2019 [1].The number is increasing in industrialized countries, including Asia, and is estimated to reach 10 million by 2050 [2,3].Although IBDs are caused by chronic inflammation, UC involves inflammation of the rectum to the small intestine, whereas CD involves inflammation of the whole gastrointestinal tract from the gut to the mouth [4].Inflammation is promoted by genetic and environmental factors such as the gut microbiome [5,6].Considering its ability to suppress inflammation, 5-aminosalicylic acid (5-ASA) is prescribed to patients with IBD as a first-line treatment [7].However, the anti-inflammatory effect of 5-ASA is insufficient for durable remission and mucosal healing [8,9].Mucosal healing, denoting the regeneration of the damaged intestinal mucosa, is essential for long-term clinical remission in patients with IBDs [10].Recently, novel therapeutics, such as biologics and small molecules, have been developed to support mucosal healing.Anti-tumor necrosis factor (TNF)-α therapies (e.g., infliximab and adalimumab) are typical biologics and are often used in patients unresponsive to 5-ASA and corticosteroid treatments [11,12].However, a certain population of patients with CD are either unresponsive or lose their response to biologics [13,14].Although 70% of patients with CD display small bowel lesions [15], the poor therapeutic efficacy of biologics in treating small bowel lesions might explain the lack of improvement in outcomes, including hospitalization [10,16,17].Another potential reason could be the low treatment efficacy of refractory perianal fistulas [18].There is no definitive evidence that new biologics and small molecules in long-term use have totally closed intestinal or anal fistula lesions in CD that cause a poor outcome and operation.Therefore, developing novel therapies for long-term clinical remission of patients with CD would be important.
Recently, stem cell transplantation therapy for IBDs has attracted attention because of its immunomodulatory function and promotion of mucosal healing [19,20].Mesenchymal stem/stromal cells (MSCs) are good cell source candidates owing to their immunosuppressive effects and ability to migrate to inflammatory sites [20][21][22][23].In the USA, EU, and Japan, darvadstrocel, a dispersion of expanded allogeneic, human adipose tissue-derived MSCs (AD-MSCs), is approved for the treatment of patients with an inadequate response to at least one conventional therapy or biologics with complex perianal fistulas with non-active or mildly active luminal CD [24].However, a recent announcement underpinned that the primary endpoint of combined remission at 24 weeks could not be achieved in the phase III ADMIRE-CD II study [25].In addition, a suitable transplantation method for MSCs, including the delivery route (i.e., intravenous, intraperitoneal, or anal injection) and cell form, has not yet been established for the treatment of IBDs [21].Therefore, further developing stem cell-based therapies and investigating transplantation methods suitable for difficult-to-treat conditions, such as complex perianal fistulas, are necessary.
MSCs are a cell population that adheres to a plastic dish under culture conditions and can be obtained from diverse tissues, such as the bone marrow, adipose tissue, placenta, dental pulp, and skin [26][27][28][29][30].Although bone marrow-derived MSCs (BM-MSCs) have been the most studied and are used in many clinical applications [31], they are present in only 0.001-0.01% of the bone marrow and exhibit invasive issues at the harvesting stage [32,33].Adipose tissues contain the MSC fraction with 1-10% stromal cells and are attracting attention as a cell source for clinical applications owing to their accessibility [34,35].Previously, we compared the percentage of the MSC fraction among subcutaneous, amnion, chorion, villus, umbilical cord, and visceral fat and demonstrated that it is the highest in subcutaneous fat [28].MSCs express specific cell-surface antigens (CD44, CD73, CD90, and CD105) and lack hematopoietic cell antigens (CD14, CD19, CD34, and CD45), endothelial marker (CD31), costimulatory molecules (CD40, CD80, and CD86), and MHC molecules (HLA class II) [26].The positive markers depend on the species and tissue of origin [27,[36][37][38][39][40][41].Considering the differences in cell-surface markers and gene expression profiles [42], the function of MSCs in migration at the inflammation site and their immunomodulatory features may vary [43,44].Additionally, it has been noted that the characterization of MSCs is altered during the clinical-scale expansion because of cellular senescence [45][46][47].
In this paper, we review clinical trials of MSCs in patients with IBDs, especially CD, from 2012 to 2023 in terms of the cell culture period for transplantation, transplantation methods, and their mode of action.Furthermore, we describe our novel findings demonstrating the advantages of isolating MSCs using fluorescence-activated cell sorting (FACS) in mouse and human studies.   2 included variations of "three-dimensional, " "organoid, " "spheroid, " "scaffold, " "spheroid-free, " "tissue engineering, " and "mesenchymal stem/stromal cells." Clinical trials were extracted from the papers searched by one author.Another author validated the list and selected reports that matched the purpose of this review.Eighteen papers of 29 cases were selected in Table 1.Complex perianal fistulas not associated with IBDs were excluded.The cases using adipose-derived stromal vascular fraction were excluded.Nineteen papers of 20 cases were selected in Table 2. MSC preparations consisting of cell sheets were excluded.

mRNA sequencing (mRNA-seq) and data reanalysis
Principal component and gene expression analyses of MSC markers were performed based on an mRNA-seq dataset from our previous study [85].The Euclidian distance was calculated based on the total genes (81,602 transcripts) after log2 transformation in the mRNA-seq dataset.Data analyses and visualizations were conducted using the RStudio environment and a Tag-Count Comparison Graphical User Interface (TCC-GUI).

Statistical analysis
All statistical analyses were performed using the statistical programming language R version 4.3.3(2024-02-29).Statistical significance was determined using the Wilcoxon rank-sum test.

Current status of clinical applications of MSCs in IBD treatment from published papers
Eighteen clinical trials for treating CD have been published in the past decade (Table 1) [48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65].Of the 18 studies, 10 used BM-MSCs, six used AD-MSCs, and two used umbilical cord-derived MSCs (UC-MSCs) as transplanted cells.These trials included four cases using MSC preparations (i.e., remestemcel-L, darvadstrocel, and TH-SC01) [55,[60][61][62].Five of the 18 studies were trials on the intravenous injection of MSCs in patients with luminal CD [48][49][50][51][52].One study used autologous BM-MSCs for intravenous injection, whereas others used allogeneic BM-or UC-MSCs.No such trials have been conducted using AD-MSCs.For the treatment of fistulizing CD, infusion into the lesion site was performed because of accessibility, with 13 of the 18 reports involving intralesional injections [54][55][56][57][58][59][60][61][62].Of the studies using intralesional injections, nine targeted perianal fistulas, two targeted peripouch or rectovaginal fistulas, and two targeted luminal CD, including strictures.One of these reports showed the serial intravenous and intralesional injections of allogeneic BM-MSCs in patients with perianal fistulizing CD [53].Three studies reported the efficacy and safety of combination therapy with autologous AD-MSCs and bioabsorbable matrices as a scaffold in intralesional injections [63][64][65].All the studies used allogeneic MSCs in intralesional injections without scaffolds.Although some trials have reported a few adverse effects, it is important to note that all studies unequivocally demonstrated the safety of MSC transplantation.Almost all studies have shown benefits regarding the efficacy of MSC therapy; however, in a few cases (i.e., patients with luminal CD), they have not shown a significant advantage.For example, Lightner et al. performed intralesional transplantation of allogeneic BM-MSCs (1.5-3.0 × 10 8 ) into four patients with luminal CD [55].They reported that the indices of clinical remission and response, with a simple endoscopic score for CD (SES-CD), dropped from 17 to 5, the Crohn's disease activity index score (CDAI) dropped from 228 to 200, and C-reactive protein (CRP) remained at 0.20-0.30.In contrast, in the control group, the SES-CD increased from 15.5 to 25.0, the CDAI increased from 146 to 158, and the CRP dropped from 3.65 to 1.50 at 3 months.No patients in the treatment group showed clinical remission or response.One patient received anti-TNF-α monoclonal antibody treatment (i.e., Cimzia) before MSC therapy.Previous studies have shown that patients with pre-treated anti-TNF-α treatment have a lower response to subsequent therapies than naive patients [86,87].Lightner et al. indicated that achieving efficacy when attempting new therapeutics for treatment-refractory patients is challenging.They also performed intralesional transplantation of allogeneic BM-MSCs (7.5 × 10 7 ) to treat peripouch fistulas in patients with ileal pouch-anal anastomosis [56].Four of 13 patients (31%) in the treated group and one of six (20%) in the control had complete clinical and radiographic healing at 6 months.Vieujean et al. showed a 40% complete stricture resolution at 48 weeks in patients with luminal CD [57].On the other hand, Molendijk et al. demonstrated the efficacy of intravenous injection of allogeneic BM-MSCs in treating refractory perianal fistulas in patients with CD [54].Low-dose administration (3 × 10 7 ) resulted in greater fistula healing than high-dose administration (9 × 10 7 ).The dose in Lightner's trial was higher than that in Molendijk's trial; this may also have caused these differences in efficacy.Thus, further analysis is needed to determine the relationship between efficacy and dose dependency on the CD condition, especially luminal CD.
The culture period of MSCs for transplantation is yet to be clearly defined.Of the 18 reports, only three clearly described the total culture period [48,51,57].Others were either partially or not described.In the papers described, the shortest culture period was 2 weeks, and       [45][46][47].Moreover, there are differences in transcriptional characteristics between cultured and freshly isolated MSCs [42].Even within the same MSC preparation, functions may differ depending on the culture period.Thus, culture days and the number of passages before transplantation should be defined for optimal efficacy.The key players in the pathogenesis of IBDs are T helper (Th) cells and regulatory T cells (Tregs) [88,89].The imbalance in these cells activates other immune cells (i.e., macrophages and B cells) exacerbating inflammation in the gut mucosa [5].Almost all studies have proposed an immunomodulatory function as a mode of action for MSC therapy.In a preclinical study, MSCs inhibited the function of Th cells and increased the population of Tregs by modulating the secretion of numerous factors, such as pro-inflammatory (e.g., interferon-γ, TNF-α, and interleukin [IL]-17) and anti-inflammatory (IL-10 and transforming growth factor-β) cytokines [90,91].Despite analyses of lymphocytes in peripheral blood and biopsy samples, the mode of action of MSCs in humans still needs to be fully understood.
Seventeen of the 19 studies used bioabsorbable scaffolds, whereas two did not.In scaffold-free trials, 3D structures were generated by expanding cultures of autologous MSCs, followed by 3D cultures in osteogenic differentiation media or media containing ascorbate [66,67].3D cultures have been performed to improve the effectiveness of bone reconstitution by facilitating osteogenesis and angiogenesis or improving adhesiveness to the cartilaginous matrix, which has been used to treat degenerative disc disease or knee chondral lesions.In contrast, in the trials with the MSC plus scaffold construct, 13 papers were trials for osteochondral and tendon regeneration, two for spinal cord and spine regeneration, one for wound healing of ulcers, and one for ischemic heart disease.Fourteen of the 19 papers used 3D constructs for bone and cartilage regeneration, indicating that surgically accessible tissues were targeted [67][68][69][70][71][72][73][74][75][76][77][78][79][80].MSCs originated from various tissues, including the bone marrow (eight reports), umbilical cord (three reports), adipose tissue (two reports), synovium (two reports), alveolar bone (two reports), gingiva (one report), and Wharton's jelly (one report).Lamas et al. reported treating patients with fullthickness rotator cuff tears using autologous BM-MSCs with type I collagen membrane [76].They used a scaffold to augment the ability of MSCs because MSCs alone are insufficient to improve the healing of repaired tendons.Morrison et al. also reported that MSCs alone cannot restore bone structure and that a supportive matrix is required [80].Depending on the tissue of origin, MSCs have different differentiation abilities [41].Zhuang et al. used BM-MSCs for treating bone defects because the MSCs can prompt attachment to the surface and inner space of porous scaffolds, such as a β-tricalcium phosphate, efficiently constructing a bioactive composite [72].Although synovium-derived MSCs have more prominent chondrogenic and less osteogenic differentiation abilities than those derived from the bone marrow or periosteum, Akgun et al. used synovium-derived MSCs with type I/ III collagen membranes to treat chondral defects of the knee [70].Apatzidou et al. reported improvements in osteoarthritis using umbilical cord blood-derived MSCs, which promoted the differentiation of endogenous chondroprogenitors through paracrine effects [78].In the case of periodontal intrabony defects, autologous gingivaderived MSCs alter the components of the extracellular matrix in gingival fibroblasts by migrating into periodontal defects [77].Thus, several trials employ MSCs to promote the multipotency of MSCs or the differentiation of endogenous cells in the lesion site rather than immunomodulatory functions; therefore, the MSCs used tend to differ depending on the type of scaffold used or the target disease.
Regarding the culture period, although eight studies did not describe the MSC expansion time, the period ranged from 14 to 50 days in the papers described.Generating 3D constructs without scaffolds requires a longer culture period (2-3 weeks or 40 days) [66,67].In contrast, when using scaffolds, the incubation period was mostly 2 days, with the shortest period being 4 h and the longest being 20-30 days.In particular, 3D cultures with osteogenic differentiation require extended culture periods.Notably, eight of the 19 reports performed in vitro 3D culture, whereas the others reported short incubation times ranging from 10 to 60 min in syringes or lacked details.Because the 3D structure of MSCs by self-aggregation enhances their immunomodulatory effects, a scaffold-free 3D MSC construct for treating inflammatory diseases, including IBDs, may be useful in clinical trials.However, evidence on the efficacy of MSC spheroids in clinical trials is currently insufficient; in the future, it is considered necessary to advance clinical research, including IBDs.

Advantages of prospective isolated MSCs using specific cell-surface markers
MSCs exhibit cellular heterogeneity with various cellsurface antigens [100].Even when similar cell-surface markers are expressed on MSCs, their functions, including differentiation potential and immunomodulatory effects, differ depending on the tissue of origin.For example, previous studies have shown that UC-MSCs have higher proliferation, colony-forming, and immunomodulation abilities than AD-MSCs [101,102].Furthermore, it is noted that adhering MSCs on plastic dishes are heterogeneous cell populations with donor-to-donor variability in clinical trials [58,103].This interindividual variability is a significant problem when autologous MSCs are used.In preclinical studies, high-quality BM-MSCs have been isolated using several specific markers by FACS (i.e., for mice, a Fig. 1 Advantage of prospective isolated CD73 + cells and their three-dimensional (3D) culture.Conventional mesenchymal stem/stromal cells (cMSCs) are a heterogeneous cell population that adheres to a plastic dish under culture conditions.CD73 + cells are a homogeneous cell population prospectively isolated using a cell sorter.Compared with cMSCs, CD73 + cells showed the downregulation of inflammatory gene expressions and the upregulation of extracellular matrix remodeling gene expressions.The 3D culture-derived spheroids of CD73 + cells (CD73 + spheroids) enhance these gene expression profiles.Additionally, CD73 + spheroids have elevated gene expressions related to Wnt signaling and immunomodulation compared with CD73.+ cells.CCL, C-C motif chemokine ligand; ECM, extracellular matrix; FN1, fibronectin 1; IL, interleukin; ITGB3, integrin β3; MGP, matrix Gla protein; MSCs, mesenchymal stem/stromal cells; POSTN, periostin; 3D, three-dimensional; TSG-6, tumor necrosis factor-α-stimulated gene 6 combination of CD140a and Sca-1; for humans, a combination of CD90, CD271, and CD146) [27,37].These prospectively isolated MSCs have a high potential for colony formation.In addition, we previously demonstrated that AD-MSCs isolated using CD73 molecules (termed CD73 + cells) have a higher colony-forming ability than conventional heterogeneous adherent MSCs (termed cMSCs) from human and rodent experiments [28].Intranasal injection of CD73 + cells reduces macrophage infiltration and suppresses fibrosis at the lesion site in mice with bleomycin-induced pulmonary fibrosis [28].Moreover, in a mouse model of dextran sulfate sodium-induced colitis, intravenous injection of CD73 + cells significantly attenuated tissue destruction compared to that observed with cMSCs [85].We also demonstrated that compared with cMSCs, human adipose tissue-derived CD73 + cells downregulated the expression of genes related to "immune response, " "inflammatory response, " and "neutrophil chemotaxis" (e.g., IL-1β, IL-6, IL-19, C-C motif chemokine ligand 2 [CCL2], CCL3, and CCL4), as revealed via a transcriptome analysis (Fig. 1) [85].In contrast, the terms for the upregulated genes were "GTP biosynthetic process, " "UTP biosynthetic process, " "negative regulation of platelet-derived growth factor receptor signaling pathway, " and "negative regulation of intrinsic apoptotic signaling pathway." These features indicate that CD73 is an ectonucleotidase that metabolizes extracellular adenosine triphosphate [104].
Our recent study also revealed upregulation of the expression of ECM-related genes (e.g., fibronectin 1, S100A13, SLC3A2, integrin β3, periostin, and FGF-1) in CD73 + cells compared with that in cMSCs (Fig. 1) [85].Furthermore, the 3D culture of CD73 + cells enhanced their characteristics, such as upregulation of the expression of ECM remodeling and Wnt signaling genes (e.g., WNT11) and downregulation of the expression of inflammatory genes (Fig. 1).Upregulation of the expression of immunomodulatory genes (e.g., matrix Gla protein and TSG-6) was also observed in 3D culture-derived CD73 + spheroids (Fig. 1).Considering these properties, CD73 + spheroids may be suitable for transanal transplantation to treat IBDs.Indeed, our preclinical study showed that CD73 + spheroids increased the engraftment rate into the lesion site compared to CD73 + cells, preventing mucosal atrophy in a mouse model of colitis.The proportion of endogenous CD140a + fibroblasts was altered after CD73 + spheroid transplantation in the lesion site.Supernatants of CD73 + spheroids, including their secretory factors, affect the gene expression profiles of fibroblasts, such as ECM remodeling and integrin downstream genes in vitro.These findings suggest that transanal transplantation of CD73 + spheroids exerts a potential therapeutic effect against IBDs via the paracrine effects of secreted factors caused by sustained engraftment.
Although the efficacy of CD73 + cells and CD73 + spheroids in treating inflammatory diseases in these animal models is promising, whether the CD73 + cell population is homogeneous remains unknown.Therefore, we reanalyzed the gene expression profiles based on transcription analysis of CD73 + cells and cMSCs.No significant differences in the expression of MSC markers, including CD73, CD44, CD90, CD105, CD146, and CD271, were observed between the CD73 + cell populations and cMSCs (Fig. 2a).Principal component analysis showed that one donor (donor #1) was distant from the others (Fig. 2b).We compared the distance based on their gene expression between CD73 + cells and cMSCs.Notably, the distance between inter-CD73 + cells was significantly smaller than that between cMSCs (P = 0.026; Fig. 2c).These results indicate that the CD73 + cell population has less donor-to-donor variability than heterogeneously adherent MSCs, which makes them clinically applicable.

Conclusions
In clinical trials of MSCs, the optimal transplantation method for target diseases must be defined; thus, the original tissue, isolation, form, and delivery route of MSCs must be adapted according to the disease.3D culture-derived MSC constructs may be suitable for treating surgically accessible bone and cartilage defects or IBDs, especially perianal fistulizing CD (Fig. 1).In addition, it is desirable to use MSCs isolated with specific cell-surface markers to avoid interindividual deviation.However, several challenges persist, such as the development of clinical antibodies for isolation and residual antibodies in transplanted MSCs; furthermore, their efficacy and safety need to be considered.Nevertheless, the properties of MSCs render them promising in regenerative medicine.

Fig. 2
Fig. 2 Characterization of homogenous CD73 + cell populations and conventional heterogeneous adherent mesenchymal stem/stromal cells (cMSCs).a Gene expression (log2) of representative MSC markers in human adipose tissue-derived CD73 + cells and cMSCs based on mRNA-seq analysis (n = 4).Statistical significance was determined using the Wilcoxon rank-sum test (P < 0.05).b Principal component analysis showing the relatedness of each sample based on the expression of the top 100 genes (n = 4 specimens).c Graph showing the interindividual Euclidian distance based on the mRNA expression (log2) of 81,602 transcripts between CD73.+ cell populations and cMSCs (n = 4 specimens).The cell culture period is approximately 14 days (less than two passages).Statistical significance was determined using the Wilcoxon rank-sum test (P < 0.05)

Table 1
Published clinical trials related to CD

Table 1 (continued) No Auto/Allo Tissue of origin No. of cells Culture period Delivery route No. of patients Efficacy Proposed mode of action Type of CD Phase Ref
Allo allogeneic, AT adipose tissue, Auto autologous, BM bone marrow, CS corticosteroid, CD Crohn's disease, CDAI Crohn's disease activity index score, HBI Harvey-Bradshaw index, IDO indoleamine 2,3-dioxygenase, i.a.intraarterial, i.l.intralesional, i.v.intravenous, MRI magnetic resonance image scan, MSC mesenchymal stem/stromal cell, NK natural killer, P passage, Treg regulatory T cells, UC ulcerative colitis, UC umbilical cord, UNK unknown cells." The search terms related to Table

Table 2
Published clinical trials using 3D MSC constructs

Tissue of origin No. of cells 3D culture/ scaffold Culture period No. of patients Efficacy Proposed mode of action Phase Ref
3D three-dimensional, UC umbilical cord, UCB umbilical cord blood, UNK unknown, VPD vertical pocket depth, VAS visual analog scales the maximum was 4 or 6 weeks in five or fewer passages.Previous studies have shown that MSCs gradually lose specific capacities such as proliferation, differentiation, and secretion during expansion Allo allogeneic, AT adipose tissue, Auto autologous, ASIA American spinal injury association, BCP biphasic calcium phosphate, BM bone marrow, β-TCP β-tricalcium phosphate, CAL clinical attachment loss, Col collagen, CT computerized tomography, ECM extracellular matrix, GA glutaraldehyde, HA hyaluronic acid, IKDC International Knee Documentation Committee, MSC mesenchymal stem/stromal cell, NSCs neural stem cells, ODI Oswestry disability index, P passage, PRP platelet-rich plasma, QOL quality of life, SD-MSCs synovium-derived MSCs, TAS Tegner activity scale,